Methods and compositions for wound healing

ABSTRACT

Assays employing fibronectin-depleted substrates are described to identify invasion inducing agents. Such agents are usefull for in vivo wound healing, including but not limited to deep wounds and chronic wounds.

This application is a continuation-in-part of U.S. application Ser. No.08/754,322 filed on Nov. 21, 1996 now U.S. Pat. No. 5,840,514.

FIELD OF THE INVENTION

The present invention relates to methods and compositions for woundhealing, and in particular, methods and compositions to promote andenhance wound healing.

BACKGROUND

The primary goal in the treatment of wounds is to achieve wound closure.Open cutaneous wounds represent one major category of wounds and includeburn wounds, neuropathic ulcers, pressure sores, venous stasis ulcers,and diabetic ulcers. Open cutaneous wounds routinely heal by a processwhich comprises six major components: i) inflammation, ii) fibroblastproliferation, iii) blood vessel proliferation, iv) connective tissuesynthesis v) epithelialization, and vi) wound contraction. Wound healingis impaired when these components, either individually or as a whole, donot function properly. Numerous factors can affect wound healing,including malnutrition, infection, pharmacological agents (e.g.,actinomycin and steroids), diabetes, and advanced age [see Hunt andGoodson in Current Surgical Diagnosis & Treatment (Way; Appleton &Lange), pp. 86-98 (1988)].

With respect to diabetes, it is known that delayed wound healing causessubstantial morbidity in patients with diabetes. Diabetes mellitus is achronic disorder of glucose metabolism and homeostasis that damages manyorgans. It is the eighth leading cause of death in the United States. M.Harris et al., “Prevalence of Diabetes and Impaired Glucose Toleranceand Glucose Levels in the US Population aged 20-40 Years,”Diabetes36:523 (1987). In persons with diabetes, vascular disease,neuropathy, infections, and recurrent trauma predispose the extremities,especially the foot to pathologic changes. These pathological changescan ultimately lead to chronic ulceration, which may necessitateamputation.

The most commonly used conventional modality to assist in wound healinginvolves the use of wound dressings. In the 1960s, a major breakthroughin wound care occurred when it was discovered that wound healing with amoist occlusive dressings was, generally speaking, more effective thanthe use of dry, non-occlusive dressings [Winter, Nature 193:293-94(1962)]. Today, numerous types of dressings are routinely used,including films (e.g., polyurethane films), hydrocolloids (hydrophiliccolloidal particles bound to polyurethane foam), hydrogels (cross-linkedpolymers containing about at least 60% water), foams (hydrophilic orhydrophobic), calcium alginates (nonwoven composites of fibers fromcalcium alginate), and cellophane (cellulose with a plasticizer) [Kannonand Garrett, Dermatol. Surg. 21:583-590 (1995); Davies, Burns10:94(1983)]. Unfortunately, certain types of wounds (e.g., diabetic ulcers,pressure sores) and the wounds of certain subjects (e.g., recipients ofexogenous corticosteroids) do not heal in a timely manner (or at all)with the use of such dressings.

Several pharmaceutical modalities have also been utilized in an attemptto improve wound healing. For example, treatment regimens involving zincsulfate have been utilized by some practitioners. However, the efficacyof these regimens has been primarily attributed to their reversal of theeffects of sub-normal serum zinc levels (e.g., decreased host resistanceand altered intracellular bactericidal activity) [Riley, Am. Fam.Physician24:107 (1981)]. While other vitamin and mineral deficiencieshave also been associated with decreased wound healing (e.g.,deficiencies of vitamins A, C and D; and calcium, magnesium, copper, andiron), there is no strong evidence that increasing the serum levels ofthese substances above their normal levels actually enhances woundhealing. Thus, except in very limited circumstances, the promotion ofwound healing with these agents has met with little success.

What is needed is a safe, effective, and interactive means for enhancingthe healing of wounds. The means should be able to be used withoutregard to the type of wound or the nature of the patient population towhich the subject belongs.

SUMMARY OF THE INVENTION

The present invention is directed at systems and methods for enhancingthe healing of wounds, especially chronic wounds (e.g., diabetic wounds,pressure sores). The compositions of the present invention are based onthe discovery that peptides containing the amino acid sequence PHSRNpromote wound healing. The present invention contemplates the use ofsuch peptides, peptide derivatives, protease-resistant peptides, andnon-peptide mimetics in the treatment of wounds.

It is not intended that the present invention be limited to the mode bywhich the compositions of the present invention are introduced to thepatient. In one embodiment, the present invention contemplates systemicadministration of the compound (e.g. intravenous). In anotherembodiment, the present invention contemplates topical administration,including but not limited to topical administration using solid supports(such as dressings and other matrices) and medicinal formulations (suchas mixtures, suspensions and ointments). In one embodiment, the solidsupport comprises a biocompatible membrane. In another embodiment, thesolid support comprises a wound dressing. In still another embodiment,the solid support comprises a band-aid.

The present invention contemplates a method for treating a wound,comprising a) providing: i) an invasion-inducing agent, and ii) asubject having at least one wound; and b) administering saidinvasion-inducing agent to said subject under conditions such that thehealing of said wound is promoted.

The present invention also contemplates a method for treating a wound,comprising a) providing: i) an invasion-inducing agent on a solidsupport, and ii) a subject having at least one wound; and b) placing thesolid support into the wound of the subject under conditions such thatthe healing of the wound is promoted.

The present invention also contemplates a method of screening candidateinvasion-inducing agents comprising: a) providing: i) inducible cells,ii) a fibronectin-depleted substrate, and iii) one or more candidateinvasion-inducing agents, b) contacting said cells in vitro with saidfibronectin-free substrate and said one or more candidateinvasion-inducing agents; and c) measuring the extent of cell invasionof said substrate. It is not intended that the present invention belimited to the type of inducible cells. In one embodiment, saidinducible cells are epithelial cells. In another embodiment, saidinducible cells are selected from the group consisting of fibroblasts,keratinocytes and muscle cells.

It is also not intended that the present invention be limited to aparticular invasion-inducing agent. In one embodiment, saidinvasion-inducing agent comprises a fibronectin-derived peptide. In apreferred embodiment, said peptide comprises the amino acid sequencePHSRN. In yet another embodiment, said peptide lacks the RGD motif. Inyet another embodiment, said peptide lacks the motif which binds theα5β1 receptor.

It is not intended that the present invention be limited by the lengthof the peptide. In one embodiment, said peptide is between five and fivehundred amino acids in length. In a preferred embodiment, said peptidecomprises the amino acids PHSRN and additional amino acids added to theamino terminus. In another embodiment, said peptide comprises the aminoacids PHSRN and additional amino acids added to the carboxy terminus. Inyet another embodiment, said peptides comprises the amino acids PHSRNand additional amino acids added to both the amino and carboxy termini.

It is not intended that the present invention be limited to specificinvasion-inducing agents. In one embodiment, the present inventioncontemplates invasion-inducing agents that comprise peptides that areprotease resistant. In one embodiment, such protease-resistant peptidesare peptides comprising protecting groups. In a preferred embodiment,endoprotease-resistance is achieved using peptides which comprise atleast one D-amino acid.

DEFINITIONS

To facilitate understanding of the invention set forth in the disclosurethat follows, a number of terms are defined below.

The term “wound” refers broadly to injuries to the skin and subcutaneoustissue initiated in different ways (e.g., pressure sores from extendedbed rest and wounds induced by trauma) and with varying characteristics.Wounds may be classified into one of four grades depending on the depthof the wound: i) Grade I: wounds limited to the epithelium; ii) GradeII: wounds extending into the dermis; iii) Grade III: wounds extendinginto the subcutaneous tissue; and iv) Grade IV (or full-thicknesswounds): wounds wherein bones are exposed (e.g., a bony pressure pointsuch as the greater trochanter or the sacrum). The term “partialthickness wound” refers to wounds that encompass Grades I-III; examplesof partial thickness wounds include burn wounds, pressure sores, venousstasis ulcers, and diabetic ulcers. The term “deep wound” is meant toinclude both Grade III and Grade IV wounds. The present inventioncontemplates treating all wound types, including deep wounds and chronicwounds.

The term “chronic wound” refers to a wound that has not healed within 30days.

The phrase “positioning the solid support in or on the wound” isintended to mean contacting some part of the wound with the solidsupport.

The phrases “promote wound healing,” “enhance wound healing,” and thelike refer to either the induction of the formation of granulationtissue of wound contraction and/or the induction of epithelialization(ie., the generation of new cells in the epithelium). Wound healing isconveniently measured by decreasing wound area.

The phrase “wound fluid contents” refers to liquid associated with awound, as well as cells, cell factors, ions, macromolecules and proteinmaterial suspended such liquid at the wound site.

The term “subject” refers to both humans and animals.

The terms “enclosure,” “compartment,” and the like refer broadly to anycontainer capable of confining a solid support within a definedlocation.

The term “solid support” refers broadly to any support, including, butnot limited to, microcarrier beads, gels, Band-Aids™ and dressings.

The term “dressing” refers broadly to any material applied to a woundfor protection, absorbance, drainage, etc. Thus, adsorbent and absorbentmaterials are specifically contemplated as a solid support. Numeroustypes of dressings are commercially available, including films (e.g.,polyurethane films), hydrocolloids (hydrophilic colloidal particlesbound to polyurethane foam), hydrogels (cross-linked polymers containingabout at least 60% water), foams (hydrophilic or hydrophobic), calciumalginates (nonwoven composites of fibers from calcium alginate), andcellophane (cellulose with a plasticizer) [Kannon and Garrett, DermatolSurg. 21:583-590 (1995); Davies, Burns10:94 (1983)]. The presentinvention specifically contemplates the use of dressings impregnatedwith the wound healing promoting and enhancing compounds of the presentinvention.

The term “biocompatible” means that there is minimal (ie., nosignificant difference is seen compared to a control), if any, effect onthe surroundings. For example, in some embodiments of the presentinvention, the dressing comprises a biocompatible membrane.

The term “peptide derivative” refers to compound having an imino group(—NH—), and more particularly, a peptide bond. Peptides may be regaredas substituted amides. Like the amide group, the peptide bond shows ahigh degree of resonance stabilization. The C-N single bond in thepeptide linkage has typically about 40 percent double-bond character andthe C═O double bond about 40 percent single-bond character.

“Protecting groups” are those groups which prevent undesirable reactions(such as proteolysis) involving unprotected functional groups. In oneembodiment, the present invention contemplates that the protecting groupis an acyl or an amide. In one embodiment, the acyl is acetate. Inanother embodiment, the protecting group is a benzyl group. In anotherembodiment, the protecting group is a benzoyl group. The presentinvention also contemplates combinations of such protecting groups.

The term “Band-Aid™” is meant to indicate a relatively small adhesivestrip comprising and adsorbent pad (such as a gauze pad) for coveringminor wounds.

DESCRIPTION OF THE DRAWINGS

FIG. 1 schematically shows the one embodiment of the substrate usedaccording to the present invention for testing invasion of cells (e.g.fibroblasts). The spatial relationship of the ectoderm of theStrongylocentrotus purpuratus embryo to its extracellular matrix and toblastocoelar structures are shown (s, spicules; h, hyalin layer; e,ectoderm; b, subectodermal basement membrane; bl, blastocoel; g, stomachof the primitive gut; c, coelomic pouches). The esophagus and intestinedo not appear on the side of the embryo shown.

FIG. 2 is a graph showing the results of the testing of fibroblasts onfibronectin-depleted substrates in vitro with and withoutinvasion-inducing agents according one embodiment of the method of thepresent invention.

FIGS. 3A and 3B are graphs showing the percentages of invaded neonatalfibroblasts, corresponding to various fragments of the plasmafibronectin cell binding domain, after placement on an invasionsubstrates. The 120 kDa and 39 kDa fragments contain the PHSRN sequence.The 11.5 kDa fragment does not. These fragments lack the α4β1 integrinbinding site in the IIICS region.

FIG. 4 is a graph presenting a dose response curve relatingconcentration of peptides containing the amino acid sequence PHSRN tofibroblast invasion into an invasion substrate.

FIG. 5 is a graph presenting a dose response curve relatingconcentration of peptides containing the amino acid sequence PHSRN tokeratinocyte invasion into an invasion substrate.

FIG. 6 is a graph presenting a dose response curve relatingconcentration of peptides containing the amino acid sequence PHSRN tohuman mammary or prostate epithelial cell invasion into an invasionsubstrate.

FIGS. 7A and 7B are graphs presenting the inductive effect of peptidescontaining the amino acid sequence PHSRN on mouse muscle satellite cellinvasion into an invasion substrate.

FIG. 8 is a graph presenting dermal wound closure data as a function oftime in genetically obese/diabetic mice, and their controls, in responseto treatment with peptides containing the amino acid sequence PHSRN.

FIG. 9 is a graph presenting dermal wound closure data as a function oftime in diabetic and non-diabetic mice treated with peptides containingthe amino acid sequence PHSRN.

FIG. 10 is a graph presenting the percentages of closed wounds as afunction of time in diabetic mice treated with peptides containing theamino acid sequence PHSRN.

GENERAL DESCRIPTION OF THE INVENTION

The therapy of wounds, particularly those which are made difficult toheal by disease, has been attempted with a variety of purified growthfactors or cytokines because these molecules can induce cellularproliferation or increase the motility of cells in wounds. Thus, ifpresented in the correct form and location at the right time, growthfactors may greatly accelerate or enhance the healing of wounds bystimulating the growth of new tissue. Given the complexity and clinicalvariability of wounds, an obvious difficulty with the application ofspecific, purified growth factors or cytokines to wounded tissue, aloneor in combination, is that their forms or specific distributions in thewound may not support their normal activities. Instead, theeffectiveness of growth factors and cytokines in promoting the healingof wounded tissue may depend on their secretion by fibroblasts ormacrophages.

The present invention contemplates a more effective approach; thisapproach involves methods that stimulate the invasion of the wound bythe cells which synthesize the growth factors and cytokines active instimulating wound repair, especially monocytes, macrophages,keratinocytes, and fibroblasts. This strategy allows the cells in theirnormal in vivo setting to secrete the active factors. This approach hasa number of advantages: (1) the temporal and spatial distributions ofthe factors are likely to be optimal because the normally active cellsin their correct settings are secreting them; (2) all the appropriatefactors are likely to be present in their active forms, irrespective ofwhether they have been identified or cloned; (3) the sequential effectsof the factors in recruiting subsequent waves of cells involved in thehealing process to the wound site are likely to be enhanced by thepresence of more initiating cells in the wound. Thus, the presentinvention can be used to augment the effects of growth factor treatment

The present invention is based on the discovery that the pure PHSRNpeptide or purified plasma fibronectin fragments (i.e.fibronectin-derived peptides) containing it, and lacking the α4β1integrin binding site in the IIICS region, are sufficient to stimulatefibroblast invasion of basement membranes in vitro under serum-freeconditions, while intact plasma fibronectin fails to stimulatefibroblast invasion. This suggests that this peptide, or forms of it notsubject to rapid proteolysis, may have similar effects on fibroblastsand monocytes/macrophages in vivo. Recruitment of fibroblasts ormonocytes/macrophages whose paracrine, regulatory effects on a varietyof neighboring cells are required for the early stages of wound healingis contemplated as a highly efficient and effective way to stimulate thecascade of regulatory interactions involved in wound healing becausethese cells will secrete the active factors or cytokines in the correcttemporal sequences and spatial locations to ensure their optimalactivities. PHSRN-containing peptides (or structurally relatedmolecules) according to the present invention stimulate the entry ofcells such as fibroblasts and monocye/macrophages into the provisionalmatrix of a wound, so that the entering cells themselves secrete thefactors and cytokines active in inducing or potentiating wound healing.

DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention contemplates 1) assays to rapidly and readilyassess both a) the invasion potential of cells of patients (such as burnor diabetes patients) as well as b) the potential of candidate inducingagents (such as non-peptide compounds having similar activity toPHSRN-containing peptides) and 2) compositions and methods for thetreatment of patients with wounds.

A. Assays for Testing Invasion Potential and Screening New Therapeutics

It may be desirable to test the potential of cells for invasion, therebypredicting the ability of a patient to respond to the wound treatmentaccording to the present invention. Similarly, it may be desirable toscreen new potential therapeutics for their level of inducing activity.Two assay systems are contemplated for such testing.

1. Fibronectin-depleted Substrates

In one assay system, the present invention contemplates usingfibronectin-depleted substrates. These are substrates that originallycontain fibronectin that are treated according to the methods of thepresent invention (see below) to remove fibronectin. It is not intendedthat the present invention be limited by the nature of the originalsubstrate; such fibronectin-containing substrates suitable for treatmentand depletion include i) complex substrates containing a variety ofextracellular proteins and ii) less complex substrates containingfibronectin along with one or two other proteins (e.g. collagen,laminin, etc.).

It is also not intended that the present invention be limited by theprecise amount of fibronectin remaining after the substrate has beentreated. In other words, while the methods of the present inventionremove fibronectin, and in some embodiments, remove substantially allfibronectin, it is within the meaning of the term “fibronectin-depleted”substrate that a small amount of fibronectin remain in the substrate.

In one embodiment, the present invention contemplates using anextracellular matrix available commercially. For example, the presentinvention contemplates treating basement membrane matrices such as ECMGEL, a matrix from mouse sarcoma (commercially available from Sigma, St.Louis, Mo.). However, it is not intended that the present invention belimited by the particular fibronectin-containig substrate. For example,other commercially available substrates are contemplated, such as thecommonly used substrate Matrigel (available from Becton DickinsonLabware, Catalog #40234); Matrigel can be treated appropriatelyaccording to the methods of the present invention so as to render it“fibronectin-depleted” (see below).

Consequently, the present invention contemplates a fibronectin-freesubstrate. In this embodiment, Matrigel is treated so that it issubstantially fibronectin-free. The preparation of fibronectin-freeMatrigel involves “panning” the Matrigel substrate on gelatin as well as“panning” the substrate on anti-fibronectin antibody (anti-humanfibronectin IgG is available commercially, such as antibody from PromegaCorporation, Madison, Wis.).

2. Naturally Occurring Fibronectin-free Substrates

In another embodiment, the present invention contemplates substratesthat are naturally free of fibronectin; such a source provides, forexample, basement membranes permeable to select types of normallyinvasive cells, such membranes being naturally serum-free. In oneembodiment, the present invention contemplates sea urchins as a sourceof such membranes. In this regard, the ectoderm of sea urchin embryos isone cell thick, and secretes an underlying basement membrane (seeFIG. 1) very similar to that of mammals. These embryos contain nocirculatory or lymphatic systems; and thus, their basement membranes areserum-free. In embryos, the subectodermal basement membrane finctionssimultaneously as a migration substrate for several, specificmesenchymal cell types while it functions as an invasion substrate forothers.

Sea urchin embryo basement membranes (SU-ECM) can be prepared by milddetergent treatment as described in D. Livant et al., CancerResearch55:5085 (1995). Briefly, adult Strongylocentrotus purpuratus seaurchins can be obtained commercially (e.g. from Pacific BioMarine), andtheir embryos cultured to the early pluteus stage in artificial seawater at 15° C. SU-ECM are then prepared from them by treatment withnonionic detergent and strerilized by dilution in the appropriate media.Cells for the invasion assay are harvested by rinsing in Hanks'balancedsalt solution, followed by brief treatment with 0.25% trypsin, 0.02%EDTA, and pelleting and resuspension in the appropriate medium with orwithout 5% FCS at a density of about 50,000 cells per ml. Whenappropriate, purified bovine plasma fibronectin (Sigma), purified 120kDa chymotryptic fragment (Gibco BRL) or PHSRN peptides (synthesized atthe Biomedical Research Core Facilities of the University of Michigan)are added to the resuspended cells prior to placement of the cells onSU-ECM. In each well of a plate used for an invasion assay, SU-ECM wereplaced in 0.5 ml of the appropriate medium, and 0.5 ml of theresuspended cells dropped on their exterior surfaces. Invasion assayswere incubated 1 to 16 hours prior to assay. If some circumstances,invasion assays were fixed in phosphate-buffered saline (PBS) with 2%formaldehyde for 5 minutes at room temperature, then rinsed into PBS.

Invasion assays are coded and scored blindly by microscopic examinationunder phase contrast at 200- and 400-fold magnification. Each cellcontacting an SU-ECM is scored for its position relative to the exterioror interior surfaces. A cell is judged to have invaded if it is locatedon an interior surface below the focal plane passing through the uppersurface of the SU-ECM, but above the focal plane passing through itslower surface. The minimum viability of the cells in each assay isalways ascertained at the time of assay by determining the fraction ofspread, adherent cells on the bottom of each well scored.

An invasion frequency is defined as the fraction of cells in contactwith basement membranes which are located in their interiors at the timeof assay. Thus, an invasion frequency of 1 denotes invasion by 100% ofthe cells in contact with basement membranes. Invasion frequencies aredetermined multiple times for each cell type assayed. For each type ofcell assayed the mean and standard deviation of the invasion frequencieswere calculated.

Regardless of which of the two types of substrates are employed, theinvasion substrates of the present invention are easy to prepare andgive rapid, highly consistent results with a variety of cells, includingbut not limited to fibroblasts and keratinocytes. While not limited toany mechanism, it is believed that cells exposed to invasion-inducingagents in this manner are potentially rendered capable of invading thesubstrate. Again, while not limited to any mechanism, it is believedthat the invasion inducing agent comprising the sequence PHSRN binds tothe α5β1 receptor on the cell and thereby induces invasion of thesubstrate. In this regard, the present invention provides a method oftreating cells comprising: a) providing i) cells expressing the α5β1receptor, ii) a fibronectin-free substrate, and iii) one or moreinvasion-inducing agents; b) culturing said cells in serum-free culturemedia on said substrate in the presence of said invasion-inducingagents; and d) measuring the extent of cell invasion of said substrate.In one embodiment, the cells are human fibroblasts.

B. Compositions and Methods for the Treatment of Patients with Wounds

It is not intended that the present invention be limited by the natureof the wound healing promoting agent. Such agents can be identifiedfunctionally by simply testing them in the above-described in vitroassays. The extent of invasion of fibroblasts (or other suitable cells)with such agents is predictive of in vivo efficacy. Thus, the presentinvention contemplates in vivo treatment with “invasion-inducingagents,” i.e. those agents which have the capability of causing invasionof cells (such as fibroblasts and keratinocytes) in the above-describedin vitro assays.

1. Peptide Derivatives

In one embodiment, the invasion-inducing agent comprises a peptidederived from fibronectin. In a preferred embodiment, said peptidecomprises the sequence PHSRN. Of course, the peptide may be larger thanfive amino acids; indeed, the peptide fragment of fibronectin maycontain hundreds of additional residues (e.g. five hundred amino acids).One such larger peptide is set forth in U.S. Pat. No. 5,492,890 (herebyincorporated by reference). In one embodiment, the PHSRN-containingpeptide is less than one hundred amino acids in length and lacks the RGDsequence characteristic of fibronectin. A variety of PHSRN-containingpeptides are contemplated, including the PHSRN peptide itself andrelated peptides where additional amino acids are added to the carboxyterminus, including (but not limited to) peptides comprising thesequence: 1) PHSRN, 2) PHSRNS, 3) PHSRNSI, 4) PHSRNSIT, 5) PHSRNSITL, 6)PHSRNSITLT, 7) PHSRNSITLTN, 8) PHSRNSITLTNL, 9) PHSRNSITLTNLT, 10)PHSRNSITLTNLTP, and 11) PHSRNSITLTNLTPG. Alternatively, PHSRN-containingpeptides are contemplated where amino acids are added to the aminoterminus, including (but not limited to) peptides comprising thesequence: 1) PEHFSGRPREDRVPHSRN, 2) EHFSGRPREDRVPHSRN, 3)HFSGRPREDRVPHSRN, 4) FSGRPREDRVPHSRN, 5) SGRPREDRVPHSRN, 6)GRPREDRVPHSRN, 7) RPREDRVPHSRN, 8) PREDRVPHSRN, 9) REDRVPHSRN, 10)EDRVPHSRN, 11) DRVPHSRN, 12) RVPHSRN, and 13) VPHSRN. Finally, thepresent invention contemplates PHSRN-containing peptides where aminoacids are added to both the amino and carboxy termini, including (butnot limited to) peptides comprising the sequencePEHFSGRPREDRVPHSRNSITLTNLTPG, as well as peptides comprising portions orfragments of the PHSRN-containing sequence PEHFSGRPREDRVPHSRNSITLTNLTPG.

Peptides containing variations on the PHSRN motif are contemplated. Forexample, the present invention also contemplates PPSRN-containingpeptides for use in the above-named assays. Such peptides may vary inlength in the manner described above for PHSRN-containing peptides.Alternatively, PPSRN may be used as a peptide of five amino acids.

Similarly, peptides comprising the sequence -HHSRN-, -HPSRN-, -PHTRN-,-HHTRN-, -HPTRN-, -PHSNN-, -HHSNN-, -HPSNN-, -PHTNN-, -HHTNN-, -HPTNN-,-PHSKN-, -HHSKN-, -HPSKN-, -PHTKN-, -HHTKN-, -HPTKN-, -PHSRR-, -HHSRR-,-HPSRR-, -PHTRR-, -HHTRR-, -HPTRR-, -PHSNR-, -HHSNR-, -HPSNR-, -PHTNR-,-HHTNR-, -HPTNR-, -PHSKR-, -HHSKR-, -HPSKR-, -PHTKR-, -HHTKR-, -HPTKR-,-PHSRK-, -HHSRK-, -HPSRK-, -PHTRK-, -HHTRK-, -HPTKR-, -PHSNK-, -HHSNK-,-HPSNK-, -PHTNK-, -HHTNK-, -HPTNK-, -PHSKK-, -HHSKK-, -HPSKK-, -PHTKK-,-HHTKK-, or -HPTKK- are contemplated by the present invention. Suchpeptides can be used as five amino acid peptides or can be part of alonger peptide (in the manner set forth above for PHSRN-containingpeptides).

As noted above, the present invention contemplates peptides that areprotease resistant. In one embodiment, such protease-resistant peptidesare peptides comprising protecting groups. In a preferred embodiment,the present invention contemplates a peptide containing the sequencePHSRN (or a variation as outlined above) that is protected fromexoproteinase degradation by N-terminal acetylation (“Ac”) andC-terminal amidation. The Ac-XPHSRNX-NH₂ peptide (which may or may nothave additional amino acids, as represented by X; the number ofadditional amino acids may vary from between 0 and 100, or more) isuseful for in vivo administration because of its resistance toproteolysis.

In another embodiment, the present invention also contemplates peptidesprotected from endoprotease degradation by the substitution of L-aminoacids in said peptides with their corresponding D-isomers. It is notintended that the present invention be limited to particular amino acidsand particular D-isomers. This embodiment is feasible for all aminoacids, except glycine; that is to say, it is feasible for all aminoacids that have two stereoisomeric forms. By convention thesemirror-image structures are called the D and L forms of the amino acid.These forms cannot be interconverted without breaking a chemical bond.With rare exceptions, only the L forms of amino acids are found innaturally occurring proteins. In one embodiment, the present inventioncontemplates PHS(dR)N-containing peptides for wound healing

2. Mimetics

Compounds mimicking the necessary conformation for recognition anddocking to the receptor binding to the peptides of the present inventionare contemplated as within the scope of this invention. For example,mimetics of PHSRN peptides are contemplated. A variety of designs forsuch mimetics are possible. For example, cyclic PHSRN-containingpeptides, in which the necessary conformation for binding is stabilizedby nonpeptides, are specifically contemplated. U.S. Pat. No. 5,192,746to Lobl, et al, U.S. Pat. No. 5,169,862 to Burke, Jr., et al, U.S. Pat.No. 5,539,085 to Bischoff, et al, U.S. Pat. No. 5,576,423 to Aversa, etal, U.S. Pat. No. 5,051,448 to Shashoua, and U.S. Pat. No. 5,559,103 toGaeta, et al, all hereby incorporated by reference, describe multiplemethods for creating such compounds.

Synthesis of nonpeptide compounds that mimic peptide sequences is alsoknown in the art. Eldred, et al, (J. Med Chem. 37:3882 (1994)) describenonpeptide antagonists that mimic the Arg-Gly-Asp sequence. Likewise,Ku, et al, (J. Med Chem. 38:9 (1995)) give further elucidation of thesynthesis of a series of such compounds. Such nonpeptide compounds thatmimic PHSRN peptides are specifically contemplated by the presentinvention.

The present invention also contemplates synthetic mimicking compoundsthat are multimeric compounds that repeat the relevant peptide sequence.In one embodiment of the present invention, it is contemplated that therelevant peptide sequence is Pro-His-Ser-Arg-Asn or Pro-Pro-Ser-Arg-Asn;in another embodiment, the relevant peptide sequence isIle-Lys-Val-Ala-Val. As is known in the art, peptides can be synthesizedby linking an amino group to a carboxyl group that has been activated byreaction with a coupling agent, such as dicyclohexylcarbodiimide (DCC).The attack of a free amino group on the activated carboxyl leads to theformation of a peptide bond and the release of dicyclohexylurea. It canbe necessary to protect potentially reactive groups other than the aminoand carboxyl groups intended to react. For example, the α-amino group ofthe component containing the activated carboxyl group can be blockedwith a tertbutyloxycarbonyl group. This protecting group can besubsequently removed by exposing the peptide to dilute acid, whichleaves peptide bonds intact.

With this method, peptides can be readily synthesized by a solid phasemethod by adding amino acids stepwise to a growing peptide chain that islinked to an insoluble matrix, such as polystyrene beads. Thecarboxyl-terminal amino acid (with an amino protecting group) of thedesired peptide sequence is first anchored to the polystyrene beads. Theprotecting group of the amino acid is then removed. The next amino acid(with the protecting group) is added with the coupling agent. This isfollowed by a washing cycle. The cycle is repeated as necessary.

In one embodiment, the mimetics of the present invention are peptideshaving sequence homology to the above-described PHSRN-containingpeptides (including, but not limited to, peptides in which L-amino acidsare replaced by their D-isomers). One common methodology for evaluatingsequence homology, and more importantly statistically significantsimilarities, is to use a Monte Carlo analysis using an algorithmwritten by Lipman and Pearson to obtain a Z value. According to thisanalysis, a Z value greater than 6 indicates probable significance, anda Z value greater than 10 is considered to be statistically significant.W. R. Pearson and D. J. Lipman, Proc. Natl Acad. Sci. (USA),85:2444-2448 (1988); D. J. Lipman and W. R. Pearson, Science,227:1435-1441 (1985). In the present invention, synthetic polypeptidesuseful in wound healing are those peptides with statisticallysignificant sequence homology and similarity (Z value of Lipman andPearson algorithm in Monte Carlo analysis exceeding 6).

3. Formulations

It is not intended that the present invention be limited by theparticular nature of the therapeutic preparation, so long as thepreparation comprises an invasion-inducing agent. For example, suchcompositions can be provided together with physiologically tolerableliquid, gel or solid carriers, diluents, adjuvants and excipients.

These therapeutic preparations can be administered to mammals forveterinary use, such as with domestic animals, and clinical use inhumans in a manner similar to other therapeutic agents. In general, thedosage required for therapeutic efficacy will vary according to the typeof use and mode of administration, as well as the particularizedrequirements of individual hosts.

Such compositions are typically prepared as liquid solutions orsuspensions, or in solid forms. Formulations for wound healing usuallywill include such normally employed additives such as binders, fillers,carriers, preservatives, stabilizing agents, emulsifiers, buffers andexcipients as, for example, pharmaceutical grades of mannitol, lactose,starch, magnesium stearate, sodium saccharin, cellulose, magnesiumcarbonate, and the like. These compositions take the form of solutions,suspensions, tablets, pills, capsules, sustained release formulations,or powders, and typically contain 1%-95% of active ingredient,preferably 2%-70%.

The compositions are also prepared as injectables, either as liquidsolutions or suspensions; solid forms suitable for solution in, orsuspension in, liquid prior to injection may also be prepared.

The invasion-inducing agents of the present invention are often mixedwith diluents or excipients which are physiological tolerable andcompatible. Suitable diluents and excipients are, for example, water,saline, dextrose, glycerol, or the like, and combinations thereof. Inaddition, if desired the compositions may contain minor amounts ofauxiliary substances such as wetting or emulsifying agents, stabilizingor pH buffering agents.

Additional formulations which are suitable for other modes ofadministration, such as topical administration, include salves,tinctures, creams, lotions, and, in some cases suppositories. For salvesand creams, traditional binders, carriers and excipients may include,for example, polyalkylene glycols or triglycerides.

EXPERIMENTAL

The following examples serve to illustrate certain preferred embodimentsand aspects of the present invention and are not to be construed aslimiting the scope thereof.

In the experimental disclosure which follows, the followingabbreviations apply: eq (equivalents); μ (micron); M (Molar); μM(micromolar); mM (millimolar); N Normal); mol (moles); mmol(millimoles); μmol (micromoles); nmol (nanomoles); g (grams); mg(milligrams); μg (micrograms); ng (nanograms); L (liters); ml(milliliters); μl (microliters); cm (centimeters); mm (millimeters); μm(micrometers); nM (nanomolar);° C. (degrees Centigrade); mAb (monoclonalantibody); MW (molecular weight); PBS (phophate buffered saline); U(units); d(days).

In some of the examples below, wounds are created in animals. Briefly,in one approach, experimental wounds were created in animals which werepre-anesthetized by inhalation of metofane and intradermal injection oflidocane. The hair on the backs of the animals was clipped and the skinwas disinfected with 70% ethanol. A piece of skin was then removed fromthe disinfected site with a 4 mm punch biopsy.

EXAMPLE 1 Production of Fibronectin-free Substrates

This example describes a purification approach for removal of plasmafibronectin (and/or cellular fibronectin) from a substrate (Matrigel).In this example, removal was attempted by affinity chromatography overGelatin-Sepharose (a technique which can be used to remove plasmafibronectin from fetal calf serum).

The Gelatin-Sepharose beads were obtained from Pharmacia (Catalog#17-0956-01). Two Kontes columns were set up with about 2 mls ofGelatin-Sepharose beads at 4 C to prevent gelling of the Matrigel. Thecolumns were then rinsed with about 10 column volumes of PBS to removethe preservative from the beads. The columns were drained to the top ofthe beads; then Matrigel was carefully added to the column. Once theMatrigel had entered the column, PBS was added to the top of the column.The Matrigel which was passed over the first column was collected andpassed over the second column. The fibronectin-depleted Matrigelcollected from the second column was plated on 48-well plates (150μl/well), sterilized under a UV light for 10 minutes and incubated at 37C overnight. The Matrigel treated in this manner failed to form a gel at37 C.

EXAMPLE 2 Production of Fibronectin-free Substrates

This example describes a purification approach for removal of plasmafibronectin (and/or cellular fibronectin) from a substrate (Matrigel).In this example, removal was attempted by successive panning on gelatin.Eight wells of 24-well plate were coated with a 2% gelatin solution (thegelatin was obtained from Becton Dickinson Labware, Catalog #11868). Thewells were filled with the gelatin solution which had been heated to 50C and incubated for 3 minutes. Then the solution was removed and thewells were allowed to air dry. Following drying, the wells werethoroughly rinsed with H₂O followed by two rinses with PBS. The plateswere again allowed to dry; thereafter they were stored at −20 C untiluse. Matrigel was thawed on ice and then added to one of the wells of agelatin-coated plate (between 800 μl and 1 ml of Matrigel was added to awell of a 24-well plate). The plate was placed in a bucket of ice in a 4C room on an orbital shaker where the Matrigel was incubated in the wellfor two hours (although overnight incubation can be used). Following theincubation, the Matrigel was moved from the first well to a second welland then incubated for two hours under the same conditions. This processwas repeated until the Matrigel had been incubated on all eight wells ofthe gelatin-coated plate.

Following the depletion of the Matrigel, it was collected in Eppendorftubes. It was then plated on a 48-well plate 150 μl/well), sterilizedunder a UV light for 10 minutes and incubated at 37 C overnight. TheMatrigel formed as gel and the following day, cells were added to eachwell.

EXAMPLE 3 Production of Fibronectin-free Substrates

This example describes a purification approach for removal of plasmafibronectin (and/or cellular fibronectin) from a substrate (Matrigel).In this example, removal was attempted by gelatin panning followed byantibody panning.

Anti-fibronectin antibody-coated wells: Wells of a 24-well plate werecoated with an anti-fibronectin antibody. A mouse monoclonal antibody tohuman fibronectin was obtained from Oncogene Science (Catalog #CP13).Each well was incubated with 1 ml of antibody at a concentration of 30μl/ml for 2 hours at room temperature. Each well was then incubated witha solution of 3% BSA in PBS for 2 hours at room temperature. Followingthe two incubation periods, the wells were thoroughly washed with PBSand stored at −20 C until use.

Depleting Matrigel of Fibronectin: Matrigel was panned over eightgelatin-coated wells (as described above in Example 2) to remove most ofthe fibronectin and its fragments. Thereafter, the Matrigel was placedin the antibody-coated wells to remove any remaining fragments offibronectin which contain the cell-binding domain but not thegelatin-binding domain The Matrigel was incubated in an ice bucket on anorbital shaker at 4 C for 2 hours. Once the Matrigel was depleted, itwas collected in Eppendorf tubes. The fibronectin-depleted Matrigel wasplated on a 48-well plate (150 μl/well), sterilized under a UV light for10 minutes and incubated at 37 C overnight. The Matrigel formed a geland the following day, cells were added to the wells.

EXAMPLE 4 Improving Gelatin Depletion as Measured by FibroblastInvasiveness

In this example, normal, neonatal fibroblasts were tested on thedepleted Matrigel material prepared according to Example 3 above (i.e.antibody depletion). As shown in FIG. 2, panning with an antibody aftergelatin depletion improved the method for removal, as measured by thereduced invasiveness of fibroblasts. On the other hand, invasiveness ofthe fibroblasts could be induced by the addition of the PHSRN peptide.The success of antibody panning suggests the feasibility of removingother components by the antibody panning methods. Other serumcomponents, such as thrombospondin, growth factors and cytokines arecontemplated by the present invention for removal by the appropriate(commercially available) antibody.

EXAMPLE 5 Conjugation of PHSRN-containing peptides

In this example, the preparation of a peptide conjugate is described.The synthetic peptide NH₂- PHSRNC can be prepared commercially (e.g.Multiple Peptide Systems, San Diego, Calif.). The cysteine is added tofacilitate conjugation to other proteins. In order to prepare a proteinfor conjugation (e.g. BSA), it is dissolved in buffer (e.g., 0.01 MNaPO₄, pH 7.0) to a final concentration of approximately 20 mg/ml. Atthe same time n-maleimidobenzoyl-N-hydroxysuccinimide ester (“MBS”available from Pierce) is dissolved in N,N-dimethyl formamide to aconcentration of 5 mg/ml. The MBS solution, 0.51 ml, is added to 3.25 mlof the protein solution and incubated for 30 minutes at room temperaturewith stirring every 5 minutes. The MBS-activated protein is thenpurified by chromatography on a Bio-Gel P-10 column (Bio-Rad; 40 ml bedvolume) equilibrated with 50 mM NaPO₄, pH 7.0 buffer. Peak fractions arepooled (6.0 ml).

The above-described cysteine-modified peptide (20 mg) is added to theactivated protein mixture, stirred until the peptide is dissolved andincubated 3 hours at room temperature. Within 20 minutes, the reactionmixture becomes cloudy and precipitates form. After 3 hours, thereaction mixture is centrifuged at 10,000×g for 10 min and thesupernatant analyzed for protein content. The conjugate precipitate iswashed three times with PBS and stored at 4° C.

EXAMPLE 6 Effect of Serum on PHSRN on Induction of Human FibroblastInvasion

In this example, the invasiveness of neonatal fibroblasts into an SU-ECMinvasion substrate is considered. Experiments were performed underserum-free conditions, or in medium with 10% fetal calf serum (FCS).Neither serum-free medium nor medium containing serum supportedfibroblast invasion. However, consistent with the induction ofmetalloproteinase gene transcription by the 120 kDa fragment of plasmafibronectin (pFn) containing the cell-binding domain, the 120 kDafragment induced fibroblast invasion in the presence or in the absenceof serum.

To insure the induction of invasion documented in these experiments wasdue to pFn sequences, and not to bound growth factors or cytokines, allof the fragments used were purified by electrophoresis on denaturinggels, followed by electoelution. Also, all fragments and sequencestested here present in solution at a molar concentration equivalent tothat of plasma fibronectin in serum. The 120 kDa cell binding domainconsists of modules 2 through 11. Modules 9 and 10 are bound by the α5β1receptor because module 9 contains the PHSRN sequence, while module 10has the RGD sequence. Accordingly, the invasion-inducing activities of agel-purified 39 kDa fragment containing modules 7-9 (and the PHSRNsequence) with a gel-purified 11.5 kDa fragment containing module 10(and the RGD) sequence was considered. As can be seen in FIGS. 3A and3B, all of the invasion-inducing activity of the plasma fibronectincell-binding domain appeared to map to the 39 kDa fragment bearingmodules 7-9 and the PHSRN sequence. To test this observation rigorously,the PHSRN peptide, which was synthesized in a peptide synthesis COREfacility, and the GRGDS peptide, which was obtained commercially, weretested in the presence or in the absence of serum for theirinvasion-inducing activities. As shown in FIGS. 3A and 3B, the PHSRNsequence contained all the invasion-stimulatory activity of the pFncell-binding domain; and the RGD sequence had no detectable activity atthe near-physiological concentrations used.

EXAMPLE 7 Dose-response Effect Between PHSRN Concentration andFibroblast Invasion.

In this example, fibroblasts were induced to invade SU-ECM byconcentrations of the PHSRN peptide ranging from 10 to 3000 ng per ml inthe presence, or in the absence of serum. As can be seen from the doseresponse curves shown in FIG. 4, the PHSRN peptide was able to inducefibroblast invasion in the presence of serum, which has been found tocontain 40 to 80 micrograms per ml of intact plasma fibronectin, and inits absence in a similar log-linear fashion. D. F. Mosher “Physiology ofFibronectin” Ann Rev. Med. 35:561 (1984).

These data suggest the metalloproteinase gene repressors produced byfibroblast α4β1 and α5β1 binding of intact plasma fibronectin do notappear to bind with such high affinity that they stop PHSRN-mediatedinvasion induction in the presence of serum. P. Huhtala et al.“Cooperative Signaling by α5β1 and α4β1 Integrins RegulatesMetalloproteinase Gene Expression in Fibroblasts Adhering toFibronectin” J. Cell Biol. 129:867 (1995). This observation isconsistent with the fact that, although induced by fibronectinfragments, fibroblast invasion in vivo must occur in the presence ofintact plasma fibronectin.

EXAMPLE 8 Induction of Keratinocyte Invasion by PHSRN in the Presence ofSerum

In this example the induction of normal keratinocyte invasion by PHSRNpeptide, in the presence of serum, is presented. Normal neonatalkeratinocytes were tested for their ability to be induced to invadeSU-ECM by the PHSRN peptide. It is notable that the profile ofkeratinocyte invasive induction into SU-ECM by PHSRN, presented in FIG.5, is similar to the profile of invasive induction of fibroblastpresented in Example 7. These data present the maximal invasionpercentages for keratinocytes at a level of about 20%. Treatment of thecells (e.g. trypsin treatment) and assay conditions (e.g. time ororientation) are likely to effect this level. In any event, it ispreferred that measurements are taken in the linear range.

EXAMPLE 9 Invasiveness of Normal Human Mammary or Prostate EpithelialCell in Response to Induction by PHSRN in a Serum ContainingEnvironrment

In this example, data is presented, FIG. 6, demonstrating that PHSRNpeptide also induces the invasive behaviors of human mammary or prostateepithelial cells. These experiments were conducted in a serum containingenvironment using SU-ECM as an invasion substrate. As with fibroblasts,immunostaining experiments showed that mammary and prostate epithelialcells express both the α5β1 and the α4β1 fibronectin receptors (notshown); thus the ability of the α5β1 receptor to bind the PHSRN sequenceon fibronectin fragments lacking the α4β1 binding site, which aregenerated in wounds may induce these epithelial cells to migrate intothe provisional matrix or into its adjacent stroma to begin woundreepithelialization.

EXAMPLE 10 Invasiveness of Mouse Muscle Satellite Cells in Response toInduction by PHSRN in a Serum Containing Environment

In this example, the ability of the PHSRN peptide to induce the invasivebehavior of a third major tissue type, muscle cells, was considered.Mouse muscle satellite cells, which function as stem cells for muscle invivo, were obtained from the laboratory of Dr. K. Kurachi (Department ofHuman Genetics). These cells were placed on SU-ECM invasion substratesin 1 microgram per ml of PHSRN peptide in the presence or absence ofserum. As shown in FIGS. 7A and 7B, PHSRN induced the invasion of SU-ECMby muscle satellite cells. Since muscle satellite cells are normallylocated inside the basement membranes surrounding the muscle fibers, indirect contact with muscle cells, and since genetically engineeredmuscle cells have so far failed to cross the basement membranesseparating them from the muscle fibers in vivo, it is interesting tospeculate that treatment with the PHSRN invasion-inducing peptide mayinduce muscle satellite cell migration into muscle in vivo, where thesecells might resume normal function.

EXAMPLE 11 In Vivo Effect of PHSRN

In this example, the effect of PHSRN on a dermal wound created ongenetically obese, diabetic mice was considered. FIG. 8 presents thedata from 10 mice wounded with 4 mm biopsy punch through skin on theback. Just after wounding, the 5 treated mice receive 5 microliters ofnormal saline containing 2 micrograms PHSRN peptide in their wounds. The5 untreated mice received 5 microliters of normal saline without PHSRN.Wound areas were measured on the days indicated by standard proceduresin which a microscope slide is placed directly on the wound and itsedges traced. The results show the rate of wound closure was acceleratedin the PHRSN-treated group as compared to the untreated controls.

EXAMPLE 12 Comparative Wound Areas

In this example, the mean wound areas in PHSRN-treated and untreatednormal and diabetic mice are considered. With respect to the diabeticmice, ten obese diabetic C57B16Ksdb/db mice received dermal wounds witha biopsy punch on day 0 according to standard methods. The wounds of 5of these mice received 2 ug of the PHSRN peptide in 5μl normal salinewithout peptide. On the days indicated, the edges of all wounds weretraced onto glass slides and the areas of the tracings determined insquare mm. This is a standard method for wound area measurement in thesemice. Six tracings of every wound were done on each day shown, and themean wound areas determined.

With respect to non-diabetic littermates, eight non-diabetic C57B16Ksdb/+ mice received duplicate dermal wounds with a biopsy punch on day 0according to standard methods. 4 to 8 mm of unwounded skin separatedeach pair of wounds. One wound on each mouse received the PHSRN peptideas described above for the diabetic mice. The other wound receivednormal saline. On the days indicated, the edges of all wounds weretraced onto glass slides for areas determination.

As shown in FIG. 9, diabetic (db/db) and normal (db/+) mice both presentratios which fall to zero. This means the treated wounds all closedprior to the untreated ones. If the PHSRN had no effect, the ratios ofthe wound areas should remain at about 1. In the alternative, if thePHSRN peptide slowed wound healing with respect to untreated mice, theratios should rise to infinity. Thus, a single application of the PHSRNpeptide to the wound shortly after wounding dramatically stimulatedwound healing in both normal and diabetic mice.

It should also be noted that the rate of wound healing in the treatedcohort of mice, relative to the untreated cohort, is promotedapproximately equally in normal and diabetic mice through day four. Thisinterval corresponds to the time of provisional matrix induction (whichrequires invasion by fibroblasts, Leukocytes, and blood vessels). Itshould be noted from these data that in the later stages of woundhealing (after the first 4 days) the diabetic mice are less responsivethan the normal mice. These data are consistent with the hypothesis thatother late-occurring processes, which are PHSRN-independent, may stilloccur more slowly in diabetic mice as compared to their normallittermates.

Expanding upon the data presented in FIG. 9, FIG. 10 presents thepercentages of completely closed wounds in PHSRN-treated and untreateddiabetic mice during the 41 day period after wounding by the abovedescribed wounding methods. Again, the results show that a singleapplication of the PHSRN peptide to the wound shortly after woundingstimulated wound healing in both normal and diabetic mice.

From the above, it should be evident that the present invention providesmethods and compositions for enhancing and promoting wound healing.Invasion-inducing agents can be readily identified using the assaysdescribed above. Thereafter, such agents can be modified or derivatizedand used therapeutically by application directly on wounds.

85 5 amino acids amino acid Not Relevant Not Relevant peptide notprovided 1 Pro His Ser Arg Asn 1 5 5 amino acids amino acid Not RelevantNot Relevant peptide not provided 2 Ile Lys Val Ala Val 1 5 6 aminoacids amino acid Not Relevant Not Relevant peptide not provided 3 ProHis Ser Arg Asn Ser 1 5 7 amino acids amino acid Not Relevant NotRelevant peptide not provided 4 Pro His Ser Arg Asn Ser Ile 1 5 8 aminoacids amino acid Not Relevant Not Relevant peptide not provided 5 ProHis Ser Arg Asn Ser Ile Thr 1 5 9 amino acids amino acid Not RelevantNot Relevant peptide not provided 6 Pro His Ser Arg Asn Ser Ile Thr Leu1 5 10 amino acids amino acid Not Relevant Not Relevant peptide notprovided 7 Pro His Ser Arg Asn Ser Ile Thr Leu Thr 1 5 10 11 amino acidsamino acid Not Relevant Not Relevant peptide not provided 8 Pro His SerArg Asn Ser Ile Thr Leu Thr Asn 1 5 10 12 amino acids amino acid NotRelevant Not Relevant peptide not provided 9 Pro His Ser Arg Asn Ser IleThr Leu Thr Asn Leu 1 5 10 13 amino acids amino acid Not Relevant NotRelevant peptide not provided 10 Pro His Ser Arg Asn Ser Ile Thr Leu ThrAsn Leu Thr 1 5 10 14 amino acids amino acid Not Relevant Not Relevantpeptide not provided 11 Pro His Ser Arg Asn Ser Ile Thr Leu Thr Asn LeuThr Pro 1 5 10 15 amino acids amino acid Not Relevant Not Relevantpeptide not provided 12 Pro His Ser Arg Asn Ser Ile Thr Leu Thr Asn LeuThr Pro Gly 1 5 10 15 18 amino acids amino acid Not Relevant NotRelevant peptide not provided 13 Pro Glu His Phe Ser Gly Arg Pro Arg GluAsp Arg Val Pro His Ser 1 5 10 15 Arg Asn 17 amino acids amino acid NotRelevant Not Relevant peptide not provided 14 Glu His Phe Ser Gly ArgPro Arg Glu Asp Arg Val Pro His Ser Arg 1 5 10 15 Asn 16 amino acidsamino acid Not Relevant Not Relevant peptide not provided 15 His Phe SerGly Arg Pro Arg Glu Asp Arg Val Pro His Ser Arg Asn 1 5 10 15 15 aminoacids amino acid Not Relevant Not Relevant peptide not provided 16 PheSer Gly Arg Pro Arg Glu Asp Arg Val Pro His Ser Arg Asn 1 5 10 15 14amino acids amino acid Not Relevant Not Relevant peptide not provided 17Ser Gly Arg Pro Arg Glu Asp Arg Val Pro His Ser Arg Asn 1 5 10 13 aminoacids amino acid Not Relevant Not Relevant peptide not provided 18 GlyArg Pro Arg Glu Asp Arg Val Pro His Ser Arg Asn 1 5 10 12 amino acidsamino acid Not Relevant Not Relevant peptide not provided 19 Arg Pro ArgGlu Asp Arg Val Pro His Ser Arg Asn 1 5 10 11 amino acids amino acid NotRelevant Not Relevant peptide not provided 20 Pro Arg Glu Asp Arg ValPro His Ser Arg Asn 1 5 10 10 amino acids amino acid Not Relevant NotRelevant peptide not provided 21 Arg Glu Asp Arg Val Pro His Ser Arg Asn1 5 10 9 amino acids amino acid Not Relevant Not Relevant peptide notprovided 22 Glu Asp Arg Val Pro His Ser Arg Asn 1 5 8 amino acids aminoacid Not Relevant Not Relevant peptide not provided 23 Asp Arg Val ProHis Ser Arg Asn 1 5 7 amino acids amino acid Not Relevant Not Relevantpeptide not provided 24 Arg Val Pro His Ser Arg Asn 1 5 6 amino acidsamino acid Not Relevant Not Relevant peptide not provided 25 Val Pro HisSer Arg Asn 1 5 28 amino acids amino acid Not Relevant Not Relevantpeptide not provided 26 Pro Glu His Phe Ser Gly Arg Pro Arg Glu Asp ArgVal Pro His Ser 1 5 10 15 Arg Asn Ser Ile Thr Leu Thr Asn Leu Thr ProGly 20 25 5 amino acids amino acid Not Relevant Not Relevant peptide notprovided 27 Pro Pro Ser Arg Asn 1 5 5 amino acids amino acid NotRelevant Not Relevant peptide not provided 28 His His Ser Arg Asn 1 5 5amino acids amino acid Not Relevant Not Relevant peptide not provided 29His Pro Ser Arg Asn 1 5 5 amino acids amino acid Not Relevant NotRelevant peptide not provided 30 Pro His Thr Arg Asn 1 5 5 amino acidsamino acid Not Relevant Not Relevant peptide not provided 31 His His ThrArg Asn 1 5 5 amino acids amino acid Not Relevant Not Relevant peptidenot provided 32 His Pro Thr Arg Asn 1 5 5 amino acids amino acid NotRelevant Not Relevant peptide not provided 33 Pro His Ser Asn Asn 1 5 5amino acids amino acid Not Relevant Not Relevant peptide not provided 34His His Ser Asn Asn 1 5 5 amino acids amino acid Not Relevant NotRelevant peptide not provided 35 His Pro Ser Asn Asn 1 5 5 amino acidsamino acid Not Relevant Not Relevant peptide not provided 36 Pro His ThrAsn Asn 1 5 5 amino acids amino acid Not Relevant Not Relevant peptidenot provided 37 His His Thr Asn Asn 1 5 5 amino acids amino acid NotRelevant Not Relevant peptide not provided 38 His Pro Thr Asn Asn 1 5 5amino acids amino acid Not Relevant Not Relevant peptide not provided 39Pro His Ser Lys Asn 1 5 5 amino acids amino acid Not Relevant NotRelevant peptide not provided 40 His His Ser Lys Asn 1 5 5 amino acidsamino acid Not Relevant Not Relevant peptide not provided 41 His Pro SerLys Asn 1 5 5 amino acids amino acid Not Relevant Not Relevant peptidenot provided 42 Pro His Thr Lys Asn 1 5 5 amino acids amino acid NotRelevant Not Relevant peptide not provided 43 His His Thr Lys Asn 1 5 5amino acids amino acid Not Relevant Not Relevant peptide not provided 44His Pro Thr Lys Asn 1 5 5 amino acids amino acid Not Relevant NotRelevant peptide not provided 45 Pro His Ser Arg Arg 1 5 5 amino acidsamino acid Not Relevant Not Relevant peptide not provided 46 His His SerArg Arg 1 5 5 amino acids amino acid Not Relevant Not Relevant peptidenot provided 47 His Pro Ser Arg Arg 1 5 5 amino acids amino acid NotRelevant Not Relevant peptide not provided 48 Pro His Thr Arg Arg 1 5 5amino acids amino acid Not Relevant Not Relevant peptide not provided 49His His Thr Arg Arg 1 5 5 amino acids amino acid Not Relevant NotRelevant peptide not provided 50 His Pro Thr Arg Arg 1 5 5 amino acidsamino acid Not Relevant Not Relevant peptide not provided 51 Pro His SerAsn Arg 1 5 5 amino acids amino acid Not Relevant Not Relevant peptidenot provided 52 His His Ser Asn Arg 1 5 5 amino acids amino acid NotRelevant Not Relevant peptide not provided 53 His Pro Ser Asn Arg 1 5 5amino acids amino acid Not Relevant Not Relevant peptide not provided 54Pro His Thr Asn Arg 1 5 5 amino acids amino acid Not Relevant NotRelevant peptide not provided 55 His His Thr Asn Arg 1 5 5 amino acidsamino acid Not Relevant Not Relevant peptide not provided 56 His Pro ThrAsn Arg 1 5 5 amino acids amino acid Not Relevant Not Relevant peptidenot provided 57 Pro His Ser Lys Arg 1 5 5 amino acids amino acid NotRelevant Not Relevant peptide not provided 58 His His Ser Lys Arg 1 5 5amino acids amino acid Not Relevant Not Relevant peptide not provided 59His Pro Ser Lys Arg 1 5 5 amino acids amino acid Not Relevant NotRelevant peptide not provided 60 Pro His Thr Lys Arg 1 5 5 amino acidsamino acid Not Relevant Not Relevant peptide not provided 61 His His ThrLys Arg 1 5 5 amino acids amino acid Not Relevant Not Relevant peptidenot provided 62 His Pro Thr Lys Arg 1 5 5 amino acids amino acid NotRelevant Not Relevant peptide not provided 63 Pro His Ser Arg Lys 1 5 5amino acids amino acid Not Relevant Not Relevant peptide not provided 64His His Ser Arg Lys 1 5 5 amino acids amino acid Not Relevant NotRelevant peptide not provided 65 His Pro Ser Arg Lys 1 5 5 amino acidsamino acid Not Relevant Not Relevant peptide not provided 66 Pro His ThrArg Lys 1 5 5 amino acids amino acid Not Relevant Not Relevant peptidenot provided 67 His His Thr Arg Lys 1 5 5 amino acids amino acid NotRelevant Not Relevant peptide not provided 68 His Pro Thr Arg Lys 1 5 5amino acids amino acid Not Relevant Not Relevant peptide not provided 69Pro His Ser Asn Lys 1 5 5 amino acids amino acid Not Relevant NotRelevant peptide not provided 70 His His Ser Asn Lys 1 5 5 amino acidsamino acid Not Relevant Not Relevant peptide not provided 71 His Pro SerAsn Lys 1 5 5 amino acids amino acid Not Relevant Not Relevant peptidenot provided 72 Pro His Thr Asn Lys 1 5 5 amino acids amino acid NotRelevant Not Relevant peptide not provided 73 His His Thr Asn Lys 1 5 5amino acids amino acid Not Relevant Not Relevant peptide not provided 74His Pro Thr Asn Lys 1 5 5 amino acids amino acid Not Relevant NotRelevant peptide not provided 75 Pro His Ser Lys Lys 1 5 5 amino acidsamino acid Not Relevant Not Relevant peptide not provided 76 His His SerLys Lys 1 5 5 amino acids amino acid Not Relevant Not Relevant peptidenot provided 77 His Pro Ser Lys Lys 1 5 5 amino acids amino acid NotRelevant Not Relevant peptide not provided 78 Pro His Thr Lys Lys 1 5 5amino acids amino acid Not Relevant Not Relevant peptide not provided 79His His Thr Lys Lys 1 5 5 amino acids amino acid Not Relevant NotRelevant peptide not provided 80 His Pro Thr Lys Lys 1 5 3 amino acidsamino acid Not Relevant Not Relevant peptide not provided 81 Arg Gly Asp1 6 amino acids amino acid Not Relevant Not Relevant peptide notprovided 82 Pro His Ser Arg Asn Cys 1 5 5 amino acids amino acid NotRelevant Not Relevant peptide not provided 83 Gly Arg Gly Asp Ser 1 5 9amino acids amino acid Not Relevant Not Relevant peptide not providedModified-site /note= “This X is a placeholder for N-terminalacetylation.” Modified-site /note= “X represents an amino group.”Modified-site /note= “The number of amino acids at this position mayvary from between 0 and 100, or more.” Modified-site /note= “The numberof amino acids at this position may vary from between 0 and 100, ormore.” 84 Xaa Xaa Pro His Ser Arg Asn Xaa Xaa 1 5 5 amino acids aminoacid Not Relevant Not Relevant peptide not provided 85 Pro Pro Ser ArgAsn 1 5

What is claimed is:
 1. A method for treating a wound, comprising: a)providing: i) an invasion-inducing agent, and ii) muscle cells, and iii)a substrate; and b) treating said muscle cells with saidinvasion-inducing agent under conditions such that said muscle cellsinvade said substrate.
 2. The method of claim 1, wherein said musclecells are muscle satellite cells.
 3. The method of claim 1, wherein saidinvasion-inducing agent is a peptide.
 4. The method of claim 3, whereinsaid peptide comprises the amino acid sequence of SEQ ID NO:
 1. 5. Themethod of claim 3, wherein said peptide is protease resistant.
 6. Themethod of claim 5, wherein said peptide comprises protecting groups. 7.The method of claim 6, wherein said peptide is N-terminally acetylatedand C-terminally amidated.
 8. The method of claim 7, wherein saidpeptide has the general formula: Ac-XPHSRNX-NH₂, as defined in SEQ IDNO: 84, wherein Ac represents the acetylated N-terminus and X representsadditional amino acids of between 0 and
 100. 9. The method of claim 3,wherein said peptide comprises at least one D-amino acid.
 10. A methodfor treating a wound, comprising: a) providing: i) an invasion-inducingagent comprising a peptide having the amino acid sequence of SEQ ID NO:1, and ii) muscle cells, and iii) a substrate; and b) treating saidmuscle cells with said invasion-inducing agent under conditions suchthat said muscle cells invade said substrate.
 11. The method of claim10, wherein said muscle cells are muscle satellite cells.
 12. The methodof claim 10, wherein said invasion-inducing agent is a peptide.
 13. Themethod of claim 12, wherein said peptide comprises the amino acidsequence of SEQ ID NO:
 1. 14. The method of claim 12, wherein saidpeptide is protease resistant.
 15. The method of claim 14, wherein saidpeptide comprises protecting groups.
 16. The method of claim 15, whereinsaid peptide is N-terminally acetylated and C-terminally amidated. 17.The method of claim 16, wherein said peptide has the general formula:Ac-XPHSRNX-NH₂, as defined in SEQ ID NO: 84, wherein Ac represents theacetylated N-terminus and X represents additional amino acids of between0 and
 100. 18. The method of claim 12, wherein said peptide comprises atleast one D-amino acid.
 19. A method for treating a wound, comprising:a) providing: i) an invasion-inducing agent comprising a peptide lackingthe RGD motif, and ii) muscle cells, and iii) a substrate; and b)treating said muscle cells with said invasion-inducing agent underconditions such that said muscle cells invade said substrate.
 20. Themethod of claim 19, wherein said peptide is protease resistant.
 21. Themethod of claim 20, wherein said peptide comprises protecting groups.22. The method of claim 21, wherein said peptide is N-terminallyacetylated and C-terminally amidated.
 23. The method of claim 22,wherein said peptide has the general formula: Ac-XPHSRNX-NH₂, as definedin SEQ ID NO: 84, wherein Ac represents the acetylated N-terminus and Xrepresents additional amino acids of between 0 and
 100. 24. The methodof claim 20, wherein said peptide comprises at least one D-amino acid.